Most cellular signaling pathways are regulated by post-translational modification of proteins, particularly phosphorylation. Reversible protein phosphorylation is found throughout eukaryotes (Hanahan et al. 2000 Cell 100: 57-70). The hallmark of many cancers is the constitutive activation of one or more of a small number of core signaling cascades including the phosphatidylinositol 3-kinase (P13K) and mitogen-activated protein (MAP) kinase pathways.
Hyperactivation of signalling pathways occurs during tumor pathogenesis as a result of over-expression of signal activators, structural alteration of kinases, or loss of negative mediators (growth factor receptor, Ras, P13K, Src, BCR-Abl, PTEN, LKB1 and SHP2; Hanahan et al. 2000 Cell 100: 57-70; McLendon et al. 2008 Nature 455: 1061-1068; Ren et al. 2005 Nat Rev Cancer 5: 172-183; Yeatman 2004 Nat Rev Cancer 4: 470-480). As a consequence, the network is rewired and a new equilibrium is established that can involve retuning sensitivity to upstream signals, bypassing routes and creation of additional nodes and connections. Cells at a later time acquire self-sufficiency in growth signals and limitless replicative potential and become insensitive to antigrowth and apoptosis signals (Hanahan et al. 2000 Cell 100: 57-70; Irish et al. 2004 Cell 118: 217-228).
For example, overexpression of epidermal growth factor receptor (EGFR) is observed in many cancers. In the case of human breast cancer, EGFR is amplified in 20-30% of the patients, and is often associated with inappropriate activation of the anti-apoptotic Ras-Raf-MEK-MAPK cascade, eventually resulting in uncontrolled cell proliferation. Ras per se is present as structurally altered forms in about 25% of human tumors, leading to constitutive activation and disengagement of this protein from the upstream mitogenic signals (Medema et al. 1993 Crit Rev Oncog 4: 615-661). One of the Ras-Raf-MAPK pathway controlled kinases, RSK, is upregulated in about 30% of all cancers and 9% of breast cancers (Barlund et al. 2000 J Natl Cancer Inst 92: 1252-1259).
Overexpression or constitutive activation of a receptor tyrosine kinase (RTK) is often a transformative event in oncogenesis (Krause et al. 2005 N Engl J Med 353: 172-187; Sebolt-Leopold et al. 2006 Nature 441: 457-462). In addition, RTK-independent activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein (MAP) kinase pathways are two of the most frequent epidemiological observations in human malignancy (i.e. phosphatase and tensin homolog (PTEN) loss of function and K-Ras gain of function, respectively). Several kinase inhibitors have been approved as drugs and more than 200 others are in development. Therefore knowledge of the genetic insult and the activation state of oncogenic kinase pathways will be crucial to proper therapy decisions.
Signaling networks in cancer cells are heterogeneous. Individual tumors derived from the same types of precursor cells may have distinct substructures within the network. In order to gain understanding of the scope of kinase signaling pathways, there is a need for a fast and convenient method to characterize not only the basal phosphorylation activities but also the manner in which protein kinases and protein phosphatases and their downstream targets perform in the pathway.